A variety of polymeric reagents including solid phase activated esters and anhydrides have been prepared for use in the derivatization of amino acids (Kalir, R., Warshawsky, A., Fridkin, M., and Patchornik, A., Eur. J. Biochem., 59, 55 (1975)., Cohen, B. J., Hafeli, H. K., and Patchornick, A., J. Org. Chem., 49, 922 (1984)., Shambhu, M. B., and Digenis, G. A., J. Chem. Soc. Chem. Commun., 619 (1974)., Martin, G. E. et al, J. Org. Chem., 43, 4571 (1978)., Martin, G. E., Shambu, M. B., and Digenis, G. A., J. Pharm. Sci., 67, 110 (1978)., Shambhu, M. B. and Digenis, G. A., Tetrahedron Lett., 18, 1627 (1973)). These polymeric reagents are, to varying degrees, structurally related to the original Merrified polymeric reagents used in the synthesis of peptides. Certain classes of polymeric activated esters and anhydrides are unique in that they are able to selectively transfer an attached moiety to mild nucleophilic species (amines, amino acids and thiols) under relatively mild reaction conditions. This moiety may then be exploited as a basis for the separation and subsequent detection of the nucleophile.
Although the synthetic organic literature contains numerous examples of the use of supported reagents for a variety of applications, examples of the use of immobilized reagents or solid phase reagents (SPR) for the derivatization of compounds of interest (analytes) for subsequent analysis by high pressure liquid chromatography (HPLC) are limited (Krull, I. S. et al, J. Liq. Chromatogr., 6, 605 (1983)., Krull, I. S. et al, J. Liq. Chromatogr., 6, 1015 (1983)., Krull, I. S. et al, J. Liq. Chromatogr., 6, 2190 (1983)., Xie, K.-H., Colgan, S. and Krull, S. I., J. Liq. Chromatogr., 6(S-2), 125 (1983)., Nondek, L., Brinkman, U. A. T., and Frei, R. W., Anal. Chem., 55, 1466 (1983)., Nondek, L., Anal. Chem., 56 1194 (1984)., Bolme, M. W. and Langer, S. H., J. Phys. Chem., 87, 3366 (1983)., Vratny, P., Ouhrabkova, J., and Copikova, J., J. Chromatogr., 191, 313 (1980)., Nondek, L., Frei, R. W., and Brinkman, U. A. T., J. Chromatogr., 282, 141 (1983)., Studebaker, J. F., J. Chromatogr., 185, 497 (1979)., Lankmayr, E. P. et al, J. Chromatogr., 224 239 (1981)).
An solid phase reagent may be prepared on a variety of solid supports which may be inorganic or polymeric in nature, including silica, glass, alumina, Florisil, or various polymeric supports derived from polystyrene, polyacrylamide or polymethacrylate. The derivatizing moiety can be physically adsorbed, intercalated, or ionically or covalently bonded to the support. There are a number of significant advantages to performing solid phase derivatizations utilizing the foregoing reagents including (1) ease of separation of the supported species from the reaction mixture, (2) reuse of a supported reagent after regeneration, (3) adaptability to continuous flow processes, (4) reduced toxicity and odor of supported species compared with low molecular weight species, and (5) chemical differences, such as solubility or altered selectivity of a reagent (resulting from steric considerations), in supported form compared with its soluble analog.
However, polymeric supports exhibit properties which render them unsuitable for continuous flow processes in that they lack dynamic stability, by shrinking or swelling as a function of solvent polarity. This limits their potential for use in derivatizations wherein the SPR is confined to a column of predetermined volume (in-line). Additionally, only macroporous polymers, which are particularly costly, exhibit sufficient rigidity for use in high pressure chromatographic systems. Finally, those polymers which have been routinely utilized for the preparation of solid phase reagents exhibit considerable hydrophobic character which can result in the irreversible adsorption of an analyte.
Particulate silica and controlled pore glass are the supports of choice when rigidity and dynamic stability are required of a particular application. However, silica and glass exhibit adsorptive properties which render them inappropriate unless further functionalized. Bonded phase silica and controlled pore glass, prepared by reaction of the inorganic support with a functionalized alkylsilane, exhibit many of the required properties. In those situations in which adsorptive secondary interactions may prove particularly detrimental, as for example in the chromatography of biological molecules, a polymeric coating of bonded phase supports further removes secondary interactions. Particulate bonded silica has been coated with polyacrylamide, polyvinylalcohol, polyethyleneimine and polysuccinimide (Alpert, A. J., J. Chromatogr., 266, 23 (1983)., Alpert, A. J., J. Chromatogr., 359, 85 (1986)).
Therefore, it is desirable that solid phase reagents be provided which exhibit sufficient dynamic stability and rigidity, and lack deletorious adsorptive properties, for use in continuous derivatizations for the purpose of qualitative and quantitative identification of analytes by high pressure liquid chromatography.